Cfse cell proliferation assay and tracking kit

T cell and CAR-T cell proliferation was detected with the CFSE Cell Proliferation Assay and Tracking Kit (Beyotime). A total of 1 × 10⁶ cells were incubated with 1 mL carboxyfluorescein succinimidyl ester (CFSE) regent at 37°C for 10 minutes. Then, 10 mL of culture medium was added and gently mixed, followed by centrifugation at 300×g for 5 minutes. Then, 5 mL medium were added, and the cells were re-centrifuged. The cells were cultured with Rg1 at different concentrations (0, 1, 5, 10, 20, and 40 μM) and proliferation was detected by flow cytometry.

T cell proliferation in the coculture was determined using Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) Cell Proliferation Assay and Tracking Kit (Beyotime, China). For CFSE staining, T cells were isolated, purified and resuspended to 1x10^6 cells/ml in 1x CFSE cell labeling solution at 37°C for 10 min, followed by centrifuging and washing with RPMI-1640 complete medium. After the coculture, CFSE-labeled T cells were collected to perform co-further culture with M2 macrophages for 72 hrs, and finally analyzed using a flow cytometer (Novocyte Quanteon). Data were interpreted as the percentage of proliferated cells.